CN1884575A - Method for constructing BAC subclone library - Google Patents

Abstract

This invention provides a kind of improved construction method of BAC subclone base. The processing steps are: extraction and purification of BAC DNA, the fragememtation and end trimming of BAC DNA; the recollection of target fragment and the attachment of vector; the transformation of attachment. For example, the BAC subclone base built by corn is with more than 1500 clones. The empty clone and coliform DNA pollution rate is lower than 5%. The inserted fragment is 3kb in average. This method is easy, cheap, fast and effective.

Description

Make up the method for BAC subclone library

One, technical field:

The present invention relates to biological technical field, relate in particular to the preparation of BAC subclone.

Two, background technology:

Organism genome sequencing meaning is not of the common run, and can obtain a large amount of genetic information by the analysis to whole genome sequence, for researchs such as functional genomics and comparative genomics lay the foundation.Measure the genome sequence of organism, identify the biological function of sequence, the evolution rule of postgraduate's object has become the heat subject in life science field.

Clone's walking method is one of sequencing of using always.Its basic link comprises: make up base and be stranded the big fragment BAC of group DNA storehouse; Utilize highdensity genetic map and BAC clone's finger printing that the BAC clone is navigated on the karyomit(e), make up full physical map of genome based on the BAC clone; BAC clone's subclone library makes up; Subclone order-checking, assembling obtain BAC and insert fragment sequence; At last, the sequence assembly with overlapping BAC clone obtains whole genome sequence.

Making up the BAC subclone library is one of important step of clone's walking method order-checking.Extensive gene order-checking requires to construct high-quality BAC subclone library, promptly has enough clone's numbers, does not have or little bacillus coli gene group DNA pollution, and empty clone is few.Generally adopt at present expensive test kit, make up the BAC subclone library, both increased and built Kucheng's basis and workload, very easily cause the pollution of DNA again, finally influence the quality of subclone library according to loaded down with trivial details working specification.Just be 30 dollars as the cost with a BAC cloned DNA of QIAGEN test kit extracting, the expense that makes up a BAC subclone library then needs about 50 dollars.Need make up 15,000 subclone library meters by the corn genome sequencing, cost is up to 750,000 dollars.At these problems, we have designed the operation steps of a series of uniquenesses, utilize the test kit of homemade cheapness, according to easy schedule of operation, just can reach the purpose that makes up high quality BAC subclone library, and expense reduces greatly, make up a subclone library and need about 15 dollars approximately.

Three, summary of the invention:

The object of the present invention is to provide a kind of easy, cheap, BAC subclone library construction process fast and efficiently.

Technical problem to be solved by this invention is: the method steps complexity of existing structure BAC subclone library is loaded down with trivial details, expense is expensive, and easily has the pollution of DNA, influences the quality of subclone library.

The present invention implements by the following technical programs:

The key link that makes up the BAC subclone library comprises: the extraction of BAC DNA and purifying; The fragmentation of BAC DNA and terminal the repairing; The recovery of target fragment and with being connected of carrier; Connect the conversion of product.And the amount of DNA and purity, high-quality BAC subclone library is very crucial for making up.

Technical solution of the present invention may further comprise the steps:

(1) extraction of BAC DNA: utilize Solution I, Solution II and Solution III (" molecular cloning laboratory manual " third edition that compound method is write referring to Sambrook and Russell), adopt alkaline lysis to prepare BAC DNA in a large number, because crude extract contains more RNA, utilize the RNase enzyme to handle crude extract, remove the RNase enzyme with the chloroform extracting, use isopropanol precipitating DNA again;

(2) purifying of BAC DNA reclaims: utilize the plain agar carbohydrate gum, low voltage, long-time electrophoresis fully separate covalently closed circle BAC DNA and bacillus coli gene group dna fragmentation.Cut superhelix BAC DNA, with sol solutions (homemade) dissolving agarose gel, simply lash BAC DNA with syringe to make it to be fractured into≤fragment of 50kb, so just, available home-made gel reclaims test kit and reclaims purifying BAC DNA, wherein, use to test kit improves, promptly be adsorbed with the centrifugal post of DNA earlier with protein liquid removal (homemade) washing, again by operation instruction rinsing and eluted dna, use this method effectively to avoid the pollution of bacillus coli gene group DNA, be recovered to capacity (rate of recovery＞85%) and purified BAC DNA;

(3) fragmentation of BAC DNA and terminal the repairing: adopt the Hydroshear  DNA boxshear apparatus of GeneMachines  company that the big fragment of BAC DNA is further sheared, the fragment of shearing concentrates between the 2.5-5kb.Be to guarantee the high efficiency of follow-up zymetology reaction, the BACDNA after utilizing home-made PCR product purification test kit to fragmentation is further purified, and has obtained the rate of recovery＞90%, purified BAC DNA, adopts the End-It of EPICENTRE ^TMThe terminal test kit of repairing of DNA is repaired BAC dna fragmentation end;

(4) recovery of target fragment and with being connected of carrier: adopt the plain agar carbohydrate gum, utilize the home-made gel to reclaim test kit, the dna fragmentation between the recovery 2.5-5kb, ligation in 4: 1 (exogenous dna fragment: ratio carrier), 16 ℃ of connections are spent the night;

(5) conversion of connection product: in order to improve transformation efficiency, to obtain enough clones, usually concentrate the DNA that connects in the product with ethanol precipitation, one of drawback of this method is very easily to cause DNA to lose, at this, utilize home-made PCR product purification test kit, effectively remove albumen and ion in the ligation liquid, the efficiently purifying concentration of DNA, electric shock transformed competence colibacillus cell (" molecular cloning laboratory manual " third edition that the competent cell preparation method writes referring to Sambrook and Russell), each conversion can obtain the clone more than 1500.

Technique effect of the present invention: inexpensive, easy and simple to handle, quick, efficient.

Four, embodiment:

Below the present invention made explanations and illustrate by the constructed embodiment of corn BAC subclone library, but protection scope of the present invention is not subjected to the restriction of specific embodiment.Can predict, the BAC subclone library that makes up different sources can be realized with reference to present embodiment.

(1) extraction of BAC DNA

1, adds 200 μ l, 2 * YT (paraxin that contains 12.5 μ g/ml) substratum in the 1.5ml pipe, inoculate corresponding BAC bacterial classification, cultivate 6hr in 37 ℃, 150rpm;

2, add 50ml 2 * YT (paraxin that contains 12.51 μ g/ml) substratum in the 250ml triangular flask, get in 1 culture 50 μ l in wherein, cultivate 14hr in 37 ℃, 250rpm;

3, use the 50ml centrifuge tube, receive bacterium with the centrifugal 10min of 6500rpm in 4 ℃;

4, nutrient solution is removed in suction, removes most raffinate in 4 ℃ with the centrifugal 1min of 6500rpm, obtains bacterial precipitation;

5, add the Solution I of 4ml precooling, thermal agitation fully scatters the mattress body;

6, add the Solution II of 8ml new system, (being no more than 5min) fast is mixed;

7, add the Solution III of 6ml precooling, be mixed gently, place 5-10min on ice;

8, in 4 ℃ with the centrifugal 15min of 11000rpm, suct clearly and newly manage in a 50ml;

9, add the 0.7V Virahol, be mixed, in 4 ℃ with the centrifugal 15min of 12000rpm;

10, abandon supernatant, and in 4 ℃ with 12000rpm centrifugal 30 seconds, remove most raffinate;

11,, and change in the 2ml centrifuge tube with 800 μ l TE dissolution precipitations;

12, add 10 μ l RNase enzymes (10mg/ml), handle 1hr with 50rpm in 37 ℃;

13, add the 1V chloroform, fully be mixed, with the centrifugal 15min of 12600rpm;

14, water intaking is newly managed in one, adds 1/10V 3MNaAc (pH5.2) and 0.7V Virahol, fully be mixed, and with the centrifugal 15min of 12600rpm;

15, abandon supernatant, and with 12000rpm centrifugal 30 seconds, remove most raffinate;

16, wash precipitation with 70% ethanol with the centrifugal 5min of 12600rpm, remove supernatant and residual;

17, room temperature is placed about 10min and is made the ethanol volatilization to the greatest extent;

18, add 160 μ l TE dissolution precipitations;

(2) purifying of BAC DNA reclaims

1, preparation 1%TAE agarose gel;

2, in the crude extract of 160 μ l, add and go up sample behind 6 * Loading Buffer;

3, low voltage (about 80V), long-time (about 3hr) electrophoresis fully separate superhelix BAC DNA and bacillus coli gene group dna fragmentation etc.;

4, cut superhelix BAC DNA, weigh;

5, add 3V sol solutions PN, dissolve fully to glue in 50 ℃;

6, lash BAC DNA once with 100 μ l syringes, make it to be fractured into≤fragment of 50kb; With the sky is the centrifugal column type sepharose DNA recovery test kit recovery purifying BAC DNA in epoch;

7, above solution is added coventional type (CB2) adsorption column,, outwell the waste liquid in the collection tube with the centrifugal 1min of 12000rpm;

8, wash adsorption column with 500 μ l protein liquid removal PD,, abandon waste liquid with the centrifugal 1min of 12000rpm;

9, wash adsorption column with 800 μ l rinsing liquid PW,, abandon waste liquid with the centrifugal 1min of 12000rpm;

10, wash adsorption column again with 500 μ l rinsing liquids,, abandon waste liquid with the centrifugal 1min of 12000rpm;

11, adsorption column is put back to collection tube,, remove rinsing liquid as far as possible with the centrifugal 2min of 12600rpm;

12, take out adsorption column, put into a clean centrifuge tube; Add 200 μ l elution buffer EB (65 ℃ of preheatings) in the middle of adsorption film, room temperature is placed 2min, and the centrifugal 1min of 12600rpm collects the BAC dna solution;-20 ℃ of preservations;

(3) fragmentation of BAC DNA and purifying

1, before the fragmentation, should guarantee that BAC DNA dissolves fully and solution in inclusion-free do not have bubble;

2, with the Hydroshear  DNA boxshear apparatus of GeneMachines  company, shear the big fragment of BAC DNA by operation instruction; This project selected parameter is: Volume (200 μ l), Speed code (12), Cycle (20); Shearing the back clip size concentrates between the 2.5-5kb;

3, with day being that the centrifugal column type PCR product purification test kit in the epoch BAC DNA after to fragmentation is further purified, to guarantee the high efficiency of follow-up zymetology reaction; Concrete operations are as follows:

4, in the BAC dna solution behind fragmentation, add 5V in conjunction with liquid PB (50 ℃ of preheatings), fully be mixed, change ultrathin type (CB1) adsorption column over to; Room temperature is placed 1min; The centrifugal 1min of 12000rpm outwells the waste liquid in the collection tube;

5, wash adsorption column with 700 μ l rinsing liquid PW, with 12000rpm centrifugal 30 seconds, abandon waste liquid;

6, wash adsorption column again with 500 μ l rinsing liquids, with 12000rpm centrifugal 30 seconds, abandon waste liquid;

7, adsorption column is put back to collection tube,, remove rinsing liquid as far as possible with the centrifugal 2min of 12600rpm;

8, take out adsorption column, put into a clean centrifuge tube; Add 40 μ l elution buffer EB (65 ℃ of preheatings) in the middle of adsorption film, room temperature is placed 1min; The centrifugal 1min of 12000rpm collects solution;

(4) end behind the fragmentation is repaired

End-It with EPICENTRE ^TMThe terminal test kit of repairing of DNA is repaired BACDNA is terminal; Concrete operations are as follows:

1, comprise in the 50 μ l reaction systems:

BAC dna solution behind the 34 μ l fragmentations

5μl 10×End-Repair?Buffer

5μl 2.5mM?dNTP?Mix

5μl 10mM?ATP

1μl End-Repair?Enzyme?Mix

2, in 16 ℃ of incubation 14hr;

3,70 ℃ of processing 10min make enzyme deactivation, termination reaction;

(5) purifying of target fragment reclaims

1, preparation 1%TAE agarose gel;

2, in 50 μ l repair liquid, add 6 * Loading Buffer, last sample, electrophoresis;

3, be the centrifugal column type sepharose DNA recovery test kit (the ultrathin adsorption column of CB1) in epoch with the sky, reclaim the dna fragmentation between the purifying 2.5-5kb; Use 20 μ l elutriant wash-outs at last;

(6) target fragment and carrier is connected

1,20 μ l ligation systems contain:

X μ l deionized water

2 μ l 10x T4DNA ligase enzyme damping fluids

2μl 50％PEG?6000

50ng pBluescriptII KS+ (EcoR V enzyme is cut/the CIP dephosphorylation)

The 200ng dna fragmentation

0.8 μ l T4DNA ligase enzyme

2, connect 14hr in 16 ℃;

3, with the sky be the concentrated and purified connection product D NA of centrifugal column type PCR product purification test kit (the ultrathin adsorption column of CB1) in epoch; Use 10 μ l elutriant wash-outs at last;

(7) conversion of connection product

In the intestinal bacteria DH10B of 20 μ l competent cell, add the concentrated and purified connection product D NA of 2 μ l; Operate by the conventional procedure that electric shock transforms.

By above process, the corn BAC subclone library of structure: clone's number surpasses 1500, and empty clone and e. coli dna pollution rate are lower than 5%, insert fragment average out to 3kb.

Claims (6)

1. the construction process of a BAC subclone library may further comprise the steps: the extraction of BAC DNA and purifying; The fragmentation of BAC DNA and terminal the repairing; The recovery of target fragment and with being connected of carrier; Connect the conversion of product.

2. the process of claim 1 wherein and comprise with protein liquid removal PD in the extraction of BAC DNA and the purifying and wash adsorption column.

3. claim 1 or 2 method, wherein the fragmentation of BAC DNA carries out with the Hydroshear  DNA boxshear apparatus by specification of GeneMachines  company.

4. claim 1 or 2 method, wherein the end of BAC DNA is repaired the End-It with EPICENTRE ^TMThe terminal test kit by specification of repairing of DNA carries out.

5. claim 1 or 2 method, carrier is wherein selected business-like pBluescriptIIKS+ carrier for use.

6. claim 1 or 2 method wherein connect product and are transformed in the intestinal bacteria DH10B competent cell.